Prodrugs of vancomycin with hydrolysis resistant polymer linkages

ABSTRACT

Vancomycin-polymer conjugates are disclosed. In preferred aspects, polymer residues which are hydrolysis resistant in vitro, are selectively attached to the sugar amino and/or N-methyl amino groups of vancomycin and related compounds. Vancomycin-polymer conjugates made by the methods and methods of treatment using the conjugates are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This patent application is a continuation of U.S. patent applicationSer. No. 10/705,740 filed on Nov. 11, 2003, which claims the benefit ofpriority from U.S. provisional patent application Nos. 60/425,890 and60/425,892, each filed Nov. 12, 2002. The contents of each applicationare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to polymeric derivatives of vancomycin.More particularly, the invention relates to select vancomycinderivatives in which the sugar amino group and/or the N-methyl aminogroup has been modified with a substantially non-antigenic polymerthrough a hydrolysis resistant linker.

BACKGROUND OF THE INVENTION

Vancomycin is an antibiotic which was initially discovered in the1950's, see U.S. Pat. No. 3,067,099. It is usually reserved for use inthe treatment of severe gram positive infections such as those caused byStaphylococcus aureus and when traditional antibiotics have failed. Overthe years, there have been several proposals for improving one or moreattributes of vancomycin, usually by continuous infusion. In anotherexample, prodrugs of vancomycin have been proposed as a way ofincreasing the solubility and circulating life of the drug.

Prodrugs include chemical derivatives of a biologically-active parentcompound which, upon administration, will eventually liberate the activeparent compound in vivo. The use of prodrugs allows the artisan tomodify one or more properties such as the onset and/or duration ofaction of a biologically-active compound in vivo. Prodrugs are oftenbiologically inert or substantially inactive forms of the activecompound. The rate of release of the active drug is influenced byseveral factors including the rate of hydrolysis of the linker whichjoins the parent biologically active compound to the prodrug carrier.

Polymer conjugates of vancomycin have also been proposed as potentialprodrugs. For example, commonly-assigned U.S. Pat. No. 6,180,095discloses benzyl elimination (BE) systems as part of a tripartitepolymer-based prodrug platform. These BE prodrug systems are designedinter alia to releasably attach polymers such as polyethylene glycol(hereinafter PEG) to hydroxyl or amine residues on small molecules.After administration to a patient, the prodrugs break down in apredictable fashion. First, the polymer portion hydrolyzes at apredictable, predetermined rate due to the presence of selectedbifunctional linkers which contain the desired “trigger”. Once thepolymer portion has been hydrolyzed, the BE system is initiated ortriggered and rapidly releases the parent compound. Commonly assignedU.S. Pat. Nos. 5,965,119 and 6,303,569 disclose related tripartateprodrug systems containing trimethyl lock triggers. Commonly assignedU.S. Patent application 60/425,892 discloses mono and di-substitutedpolymeric prodrugs employing releasably linked platforms. Commonlyassigned U.S. Pat. No. 6,395,266 discloses a branched polymeric platformsystem useful for multiple loading of biologically active moieties.Commonly assigned U.S. Pat. Nos. 6,127,355 and 5,965,566 disclose highmolecular weight polymer conjugates as drug delivery systems. Commonlyassigned U.S. Patent Applications 60/425,890 and 60/425,892 disclosemethods of making releasable polymeric vancomycin derivatives. In theaforementioned applications, the polymer platform is attached at eitherone or both of the sugar amine group and the N-methyl amine group of thevancomycin or vancomycin derivative. The disclosure of each of theabove-mentioned commonly-assigned patents and applications isincorporated herein by reference.

In spite of the fact that vancomycin is listed among the variousbiologically active compounds having an available amino group forattachment of the prodrug platform in each of the foregoingcommonly-assigned patents, further advances have been sought to refineand improve vancomycin therapies. In the past, it was thought that usingpermanent bonds, i.e. those substantially resistant to hydrolysis, toattach the vancomycin to a polymer such as PEG would not providesufficiently active compounds. It has now been discovered thatattachment of polymeric platforms through amide, urea, carbamate orother similar hydrolysis-resistant bonds is advantageous and can provideadditional prodrugs. The vancomycin prodrugs of the present invention,have been shown to hydrolyze and liberate vancomycin in vivo over anextended period of time. Advantages of these types of prodrugs include,extended circulating life due to very slow hydrolysis of the polymerportion to release native drug and a more efficient cost effectivemanufacturing process. New versions of prodrugs capable of maintainingefficacy and reducing negative side effects while maintaining costeffectiveness is an ongoing need. This invention addresses such needs.

SUMMARY OF THE INVENTION

In one aspect of the invention, a compound of the formula (I) isprovided:

wherein:

R₃-R₅ are each independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆alkenyls, C₃₋₁₂ branched alkenyls, C₁₋₄ alkynyls, C₃₋₁₂ branched alkyls,C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxyalkyl,phenoxyalkyl and C₁₋₆ hetero-alkoxys;

R₆ is OH, NH-aryl, NH-aralkyl, or NH—C₁₋₁₂ alkyl;

w is 1 or 2;

Q_(a) is H or a residue of the formula:

wherein:

R₁ is a polymer residue;

Y₁ is O, S or NR₅, and

L₁ is a hydrolysis resistant bifunctional linker;

q is 0 or a positive integer; and

Q_(b) is H or a residue of the formula:

wherein:

R₂ is a polymer residue;

Y₂ is O, S or NR₅, and

L₂ is a hydrolysis resistant bifunctional linker;

s is 0 or a positive integer;

provided that Q_(a) and Q_(b) are both not simultaneously H.

In others aspect of the invention the polymeric residue (R₁) or (R₂)includes both an alpha and an omega terminal linking group so that twoequivalents of the vancomycin residue are delivered. An example of sucha bifunctional polymer conjugate is illustrated below as formulas (II)and (III):

(i)-R¹-(i)  (II)

and

(ii)-R₂-(ii)  (III)

wherein

(i) is

(ii) is

wherein all variables are as described above.

Another aspect of the invention includes methods of treating vancomycinsusceptible diseases in mammals, i.e. a human, comprising administeringa compound of the formula (I) to a mammal in need of such treatment,whereby, the vancomycin-based compound of formula (I) undergoesdegradation and releases vancomycin or vancomycin derivative in vivo.

For purposes of the present invention, the term “residue” shall beunderstood to mean that portion of a vancomycin compound or bifunctionallinker which remains after it has undergone a substitution reaction.

For purposes of the present invention, the term “polymer residue” or“PEG residue” shall each be understood to mean that portion of thepolymer or PEG which remains after it has undergone a reaction with avancomycin compound such as those described herein as being of formula(I).

For purposes of the present invention, the term “alkyl” shall beunderstood to include straight, branched, substituted, e.g. halo-,alkoxy-, nitro-, C₁₋₁₂ alkyls C₃₋₈ cycloalkyls or substitutedcycloalkyls, etc. Positive integer shall mean an integer greater than orequal to one, preferably between 1 and 10 and more preferably 1.

For purposes of the present invention, the term “substituted” shall beunderstood to include adding or replacing one or more atoms containedwithin a functional group or compound with one or more different atoms.

For purposes of the present invention, substituted alkyls includecarboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls andmercaptoalkyls; substituted alkenyls include carboxyalkenyls,aminoalkenyls, dialkenylaminos, hydroxyalkenyls and mereaptoalkenyls;substituted alkynyls include carboxyalkynyls, aminoalkynyls,dialkynylaminos, hydroxyalkynyls and mercaptoalkynyls; substitutedcycloalkyls include moieties such as 4-chlorocyclohexyl; aryls includemoieties such as napthyl; substituted aryls include moieties such as3-bromo-phenyl; aralkyls include moieties such as toluoyl; heteroalkylsinclude moieties such as ethylthienyl; substituted heteroalkyls includemoieties such as 3-methoxy-thienyl; alkoxy includes moieties such asmethoxy; and phenoxy includes moieties such as 3-nitrophenoxy.Halo-shall be understood to include fluoro, chloro, iodo and bromo.

The term “sufficient amounts” for purposes of the present inventionshall mean an amount which achieves a desired effect or therapeuticeffect as such effect is understood by those of ordinary skill in theart.

One advantage of the compounds of the invention is that vancomycinconjugate has an extended circulating life. There is also provided ameans for controlling the rate of hydrolysis of the derivative. Thus,the artisan has the ability to include varied substituents that allowfor modulation of the rate of hydrolysis of the prodrug to optimize thepK profile, reduce dose frequency and its related medical costs. Themodifications described herein also allow one to maintain serum levelsand prevent bacterial resistance of vancomycin from developing.

The conjugates made by the methods of the invention also provide aneconomic advantage in the manufacturing process using high molecularweight polymers.

Other and further advantages will be apparent from the followingdescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 schematically illustrate methods of forming compounds of thepresent invention which are described in the Examples.

DETAILED DESCRIPTION A. Formula (I)

In one aspect of the invention compounds of the formula (I) areprovided:

wherein:

R₃-R₅ are each independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloakyls, C₁₋₄ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆alkenyls, C₃₋₁₂ branched alkenyls, C₁₋₆ alkynyls, C₃₋₁₂ branchedalkynyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆alkoxyalkyl, phenoxyalkyl and C₁₋₆ hetero-alkoxys;

R₆ is OH, NH-aryl, NH-aralkyl, or NH—C₁₋₁₂ alkyl;

w is 1 or 2;

Q_(a) is H or a residue of the formula:

wherein:

R₁ is a polymer residue;

Y₁ is O, S or NR₅;

L₁ is a hydrolysis resistant bifunctional linker;

q is 0 or a positive integer; and

Q_(b) is H or a residue of the formula:

wherein:

R₂ is a polymer residue;

Y₂ is O, S or NR₅;

L₂ is a hydrolysis resistant bifunctional linker; and

s is 0 or a positive integer;

provided that Q_(a) and Q_(b) are both not simultaneously H.

In another aspect of the invention R₁ can further comprise a cappinggroup J selected from the group consisting of OH, NH₂, SH, CO₂H, C₁₋₆alkyl moieties, and a compound of the formula (i),

In another aspect of the invention R₂ can further comprise a cappinggroup J selected from the group consisting of OH, NH₂, SH, CO₂H, C₁₋₆alkyl moieties, and a compound of the formula (II),

wherein all variable are as described above.

This aspect of the invention allows for bifunctional compounds that areformed when the polymeric residue (R₁) or (R₂) includes both an alphaand an omega terminal linking group so that two equivalents of thevancomycin residue is delivered. An example of such a bifunctionalpolymer conjugate is illustrated below as formulas (II) and (III):

(i)-R₁-(i)  (II)

and

(ii)-R₂-(ii)  (III)

wherein (i) is

(ii) is

wherein all variables are as described above.For the compounds of Formula (I) the following are preferred,

Y₁ and Y₂ are independently O;

R₃ and R₄ are each independently selected from among hydrogen, or CH₃;

R₆ is OH or NH-aryl;

q and s are independently 0-2, and

w is 1.

While the above formula (I) covers many of the more well knownvancomycin-type compounds known to have biological activity, it is to beunderstood that the invention embraces not only these specificcompounds, but also those vancomycin-based compounds know to artisans ofordinary skill to have a sugar amino group. For example, the inventiveprocesses described herein can also be carried out with the vancomycinderivatives described in, for example, EP 0 201 251, “The Role ofHydrophobic Substituents in the Biological Activity of GlycopeptideAntibiotics”, J. Am. Chem. Soc. 2000, 122, 12608-12609 and U.S. Pat.Nos. 4,495,179, 3,067,099, 4,556,008, 4,548,925 and 4,547,488 to namebut a few. The disclosure of each of the foregoing is incorporatedherein by reference. In most preferred aspects of the invention,however, the vancomycin compound employed for the processes describedherein is of the formula (V)

B. Polymer Residues

As stated above, R₁ and R₂ are polymer residues. Preferably R₁ and R₂are water soluble linear, branched or multiple arm polymer residueswhich are preferably substantially non-antigenic such as polyalkyleneoxide (PAO's) and more preferably polyethylene glycol. For purposes ofillustration and not limitation, the linear polyethylene glycol (PEG)residue portion of R₁ and R₂ can be selected from among:

J-O—(CH₂CH₂O)_(x)—

J-O—(CH₂CH₂O)_(x)—CH₂C(O)—O—,

J-O—(CH₂CH₂O)_(x)—CH₂CH₂NR₇—,

J-O—(CH₂CH₂O)_(x)—CH₂CH₂SH—,

—OC(O)CH₂—O—(CH₂CH₂O)_(X)—CH₂C(O)—O—,

—NR₇CH₂CH₂—O—(CH₂CH₂O)_(X)—CH₂CH₂NR₇— and

—SHCH₂CH₂—O—(CH₂CH₂O)_(X)—CH₂CH₂SH—.

wherein:

x is the degree of polymerization;

R₇ is selected from among hydrogen, C₁₋₆ alkyls, C₂₋₆ alkenyls, C₂₋₆alkynyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, substituted alkenyls, C₂₋₆ substituted alkynyls, C₃₋₈substituted cycloalkyls, aryls substituted aryls, aralkyls, C₁₋₆heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxyalkyl,phenoxyalkyl and C₁₋₆ heteroalkoxy, and J is a capping group asdescribed above with regard to formulas (II) and (III).

For illustrative purposes, in the case of multiple arm polyethyleneglycol (PEG) residues, examples of possible conjugates are selected fromamong:

wherein

m is 0-4;

z is 0 or 1;

L₄ is the same as that which defines L₁₋₃;

D is a moiety of the formula V_(a) or V_(b);

R₁′=

—(CH₂CH₂O)_(x)—;

—(CH₂CH₂O)_(X)—CH₂C(O)—;

—(CH₂CH₂O)_(X)—CH₂CH₂NR₇—, and

—(CH₂CH₂O)_(X)—CH₂CH₂SH—;

-   -   wherein x is a positive integer;

R₁₃₋₂₄ are independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls C₁₋₆ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆alkenyls, C₃₋₁₂ branched alkenyls, C₁₋₆ alkynyls, C₃₋₁₂ branchedalkynyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ hetero-alkyls, C₁₋₆alkoxyalkyl, phenoxyalkyl and C₁₋₁₆ heteroalkoxys;

V_(a) is

V_(b) is

wherein, d, e, q, and S are each independently 0 or 1, and all othervariables are as described above.

Preferably, R₁₃₋₂₄ are independently selected from among hydrogen andC₁₋₆ alkyls; n is 1-400, more preferably 50-250, and m is 0-20, morepreferably 0-4.

The multiple arm polymeric residues, allow for multiple points ofattachment thereby increasing the loading of the vancomycin orvancomycin derivative residues.

In one particularly preferred embodiment, R₁ and R₂ are selected fromamong

CH₃—O—(CH₂CH₂O)_(x)—,CH₃—O—(CH₂CH₂O)_(x)—CH₂C(O)—O—,

CH₃—O—(CH₂CH₂O)_(X)—CH₂CH₂NH— and CH₃—O—(CH₂CH₂O)—CH₂CH₂SH—,

where x is a positive integer, selected so that the total weight averagemolecular weight is from about 5,000 to about 100,000 Da. Preferably,the total weight average molecular weight is from about 10,000 to about80,000 Da, with from about 20,000 to about 40,000 Da being morepreferred. The most preferable total Molecular weight of the polymer is40,000 Da depending upon the needs of the artisan.

In a more preferred embodiment, in the case of multiple arm polyethyleneglycol (PEG) residues, possible conjugates are selected from among:

wherein

-   -   R₁₃₋₂₄ are independently selected from among hydrogen, and C₁₋₄        alkyls;    -   z is 0 or 1;    -   n is 50-250;    -   m is 0-4; and    -   D is V_(a) or V_(b) as described above.

Preferably, the total molecular weight range of the multiple arm polymerresidue is 20,000 Da to 40,000 Da.

Also contemplated within scope of the invention are terminally branchedpolymer conjugates such as those compounds described in commonlyassigned PCT publication numbers WO02/065988 and WO02/066066, thedisclosure of each being incorporated herein by reference. Within thesegeneral formulae, the following are preferred:

where R is a linear polymeric residue such as those described above forR₁ and R₂, and B is a moiety of the formula:

wherein

-   -   L₃ is the same as that which describes L₁ and L₂;    -   o is 0 or 1; and    -   D is a moiety of formula V_(a) or V_(b)

Additionally, branched polymer residues such as those compoundsdescribed in commonly assigned U.S. Pat. Nos. 5,643,575; 5,919,455 and6,113,906, the disclosure of each being incorporated herein by referenceare also contemplated within the scope of the invention. These residuesallow for the following preferred conjugates to be formed:

wherein:

-   -   (a) is an integer of from about 1 to about 5;    -   Z is O, NR₈, S, SO or SO₂; where R₈ is H, C₁₋₈ alkyl, C₁₋₈        branched alkyl, C₁₋₈ substituted alkyl, aryl or aralkyl;    -   (m) is 0 or 1;    -   (p) is a positive integer, preferably from about 1 to about 6,        and    -   D is a moiety of formula V_(a) or V_(b).

PEG is generally represented by the structure:

and R₁ and R₂ preferably comprise residues of this formula.

The degree of polymerization for the polymer (x) can be from about 10 toabout 2,300. This represents the number of repeating units in thepolymer chain and is dependent on the molecular weight of the polymer.The (J) moiety is a capping group as defined herein, i.e. a group whichis found on the terminal of the polymer and, in some aspects, can beselected from any of NH, OH, SH, CO₂H, C₁₋₆ alkyls or other PEG terminalactivating groups, as such groups are understood by those of ordinaryskill.

Branched PEG derivatives such as those described in commonly-assignedU.S. Pat. No. 5,643,575 (the 1575 patent), “star-PEG's” and the“multi-armed PEGs” such as those shown above and described in NektarCorporation's 2003 catalog “Polyethylene Glycol and Derivatives forAdvanced PEGylation” are useful in the methods of the invention. Thedisclosure of each of the foregoing is incorporated herein by reference.The branching afforded by the '575 patent allows secondary or tertiarybranching as a way of increasing polymer loading on a biologicallyactive molecule or enzyme from a single point of attachment. It will beunderstood that the water-soluble polymer can be functionalized forattachment to the bifunctional linkage groups if required without undueexperimentation.

Although PAO's and PEG's can vary substantially in weight averagemolecular weight, preferably, R₁ and R₂ have a total weight averagemolecular weight of from about 20,000 to about 40,000 Da in most aspectsof the invention.

The polymeric substances included herein are preferably water-soluble atroom temperature. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained.

In a further embodiment, and as an alternative to PAO-based polymers, R₁and R₂ are optionally selected from among one or more effectivelynon-antigenic materials such as dextran, polyglutamic acid, polyasparticacid, polyhydroxyethyl-aspartate (poly-HEA), chitans, polyvinylalcohols, carbohydrate-based polymers, hydroxypropylmethylacrylamide(HPMA), polyalkylene oxides, and/or copolymers thereof. See alsocommonly-assigned U.S. Pat. No. 6,153,655, the contents of which areincorporated herein by reference. It will be understood by those ofordinary skill that the same type of activation is employed as describedherein as for PAO's such as PEG. Those of ordinary skill in the art willfurther realize that the foregoing list is merely illustrative and thatall polymeric materials having the qualities described herein arecontemplated and that other polyalkylene oxide derivatives such as thepolypropylene glycols, etc. are also contemplated.

C. Hydrolysis Resistant Linkers

In many aspects of the invention, and formula (I) in particular, L₁₋₃are hydrolysis resistant bifunctional linking groups which facilitateattachment of the vancomycin or vancomycin derivative to the polymerstrands, i.e. R₁ and R₂. The linkage provided can be either direct orthrough further coupling groups known to those of ordinary skill. Inthis aspect of the invention, L₁₋₃ can be selected from among:

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)—

—[C(O)]_(v)(CR₂₆R₂₇)_(t)—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇O)_(t)(CR₂₈R₂₉)_(y)O—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇O)_(t)(CR₂₈R₂₉)_(y)—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)O—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)(CR₂₈CR₂₉O)_(y)NR₃₀—

—[C(O)]_(v)O(CR₂₆R₂₇)_(t)NR₃₀—

—[C(O)]_(v)O(CR₂₆R₂₇)_(t)O—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)NR₃₀—

—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)(CR₂₈CR₂₉O)_(y)—

—[C(O)]_(v)NR₂₅(CR₂₆CR₂₇O)_(t)—

—[C(O)]_(v)NR₂₅(CR₂₆CR₂₇O)_(t)(C₂₈R₂₉)_(y)NR₃₀—

—[C(O)]_(v)NR₂₅(CR₂₆CR₂₇O)_(t)—

—[C(O)]_(v)O(CR₂₆R₂₇)_(t)—NR₃₀—

—[C(O)]_(v)—O(CR₂₆R₂₇)_(t)NR₃₀—

—[C(O)]_(v)O(CR₂₆R₂₇)_(t)O—

—[C(O)]_(v)O(CR₂₆CR₂₇O)_(t)NR₃₀—

wherein:

R₂₅-R₃₀ are independently selected from the group consisting ofhydrogen, C₁₋₆ alkyls, C₂₋₆ alkenyls, C₂₋₆ alkynyls, C₃₋₁₉ branchedalkyls, C₃₋₈ cycloakyls, C₁₋₆ substituted alkyls, C₂₋₆ substitutedalkenyls, C₂₋₆ substituted alkynyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls, substitutedC₁₋₆hetero-alkyls, C₁₋₆ alkoxyalkyl, phenoxyalkyl and C₁₋₆hetero-alkoxys; and

R₃₁ is selected from the group consisting of hydrogen, C₁₋₆ alkyls, C₂₋₆alkenyls, C₂₋₆ alkynyls, C₃₋₁₉ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₂₋₆ sub-stituted alkenyls, C₂₋₆ substitutedalkynyls, C₃₋₈ substituted cyclo-alkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ hetero-alkyls, C₁₋₆alkoxy-alkyl, phenoxyalkyl and C₁₋₆ heteroalkoxys, NO₂, haloalkyl andhalogen;

t and y are individually selected positive integers, preferably fromabout 1 to about 4, and

v is 0 or 1.

In other aspects of the invention, L₁₋₃ can independently include anamino acid residue. The amino acid can be selected from any of the knownnaturally-occurring L-amino acids, e.g., alanine, valine, leucine,isoleucine, glycine, serine, threonine, methionine, cysteine,phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid,lysine, arginine, histidine, proline, and/or a combination thereof, toname but a few.

The amino acid residues are preferably of the formula

wherein X′ is O, S or NR₉, Y₅ is O, S or NR₁₀, and R₉, R₁₀ and R₁₁ areindependently selected from the same group as that which defines R₃ buteach is preferably H or lower alkyl (i.e. C₁₋₆ alkyl); and f is apositive integer from about 1 to about 10, preferably 1.

Desirable amino acid residues include all of the knownnaturally-occurring L-amino acids. For example, L-isoleucine as atransport enhancer is exemplified in the Examples provided below.Surprisingly, it has also been discovered that D-amino acids are usefulas transport enhancers, e.g., both D and L-alanine, and other analogousamino acid optical isomers, show the same activity. Derivatives andanalogs of the naturally occurring amino acids, as well as variousart-known non-naturally occurring amino acids (D or L), hydrophobic ornon-hydrophobic, are also contemplated to be within the scope of theinvention. Simply by way of example, amino acid analogs and derivatesinclude: 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid,2,4-diaminobutyric acid, desmosine, 2,2-diaminopimelic acid,2,3-diaminopropionic acid, n-ethylglycine, N-ethylasparagine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, sarcosine, N-methylisoleucine, 6-N-methyllysine,N-methylvaline, norvaline, norleucine, ornithine, and others toonumerous to mention, that are listed in 63 Fed. Reg. 29620, 29622,incorporated by reference herein.

D. Leaving or Activating Groups

Where mentioned with regard to the synthesis of the polymer conjugatesdescribed herein, suitable leaving groups or activating groups include,without limitations, moieties such as N-hydroxybenzotriazolyl, halogen,N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl,N-hydroxysuccinimidyl; thiazolidinyl thione, or other good leavinggroups as will be apparent to those of ordinary skill. For purposes ofthe present invention, leaving groups are to be understood as thosegroups which are capable of reacting with an amino group (nucleophile)found on the vancomycin compound.

E. Synthesis of Polymer Conjugates

Synthesis of specific representative polymer prodrugs is set forth inthe Examples. Generally, however, in one preferred method of preparing aprodrug of the invention, the vancomycin-polymer conjugates are preparedby reacting a vancomycin compound such as, for example, a compound offormula (V) as shown above with a polymer residue i.e. a linear,terminally branched or multi-arm polymer residue, that contains at leastone leaving group capable of reacting with the sugar (V₃) amino group ofa vancomycin compound. This is done in the presence of at least about a20-fold molar excess of triethylamine (TEA) and a sufficient amount ofdimethylformamide (DMF). The ratio of vancomycin to the polymer residueis based on the amount of leaving groups present on the branchedpolymer. Preferably, there is at least about a 1:1 ratio of vancomycinto leaving groups.

Important aspects of this embodiment are the selection and amount of thebase used in the reaction of the vancomycin compound with the activatedpolymer, e.g. the polymer residue containing the leaving group. Sincethe vancomycin compounds employed in the invention usually contain twoamino groups, care must be taken during the reaction so as to avoidformation cross-linked conjugates and/or heterogeneous mixtures ofvancomycin-polymer conjugates in which the polymer termini are attachedat more than one of the sugar amino (V₃) and N-methyl amino (X₁). It hasbeen surprisingly found that when the preferred amount of at least about20 equivalents of TEA in combination with a sufficient amount of DMF inthe presence of a sufficient amount of 4 Å molecular sieves is used, itis possible to obtain a substantially homogeneous reaction product of apolymer residue containing a vancomycin compound attached to eachterminal end thereof via the vancomycin V₃ amino group. In preferredaspects of the method, at least about 10 equivalents of TEA are used inthe reaction while in more preferred aspects, at least about 20equivalents are used most preferably 40 equivalents of TEA are used.

For purposes of the present invention, the amount of the solvent DMFemployed in the reaction is referred to as a “sufficient amount”. Aswill be appreciated by those of ordinary skill, this amount will besufficient to dissolve the reactants. In most aspects of the invention,the amount of DMF employed will range form about 10 mL/g to about 500mL/g and preferably from about 100 mL/g to about 200 mL/g based upon thevancomycin compound used.

In another aspect of the invention there are provided higher payload,polymer-vancomycin conjugates in which the termini of the polymerresidue are attached to the X₁ or N-methyl amino group of the vancomycincompound. Such compounds can be formed by capping the V₃ amino group ofa vancomycin compound of formula (I) and thereafter reacting the V₃capped vancomycin compound with an activated polymer containing at leastone leaving group capable of reacting with the N-methyl-amino group ofthe vancomycin compound under conditions sufficient to form polymerconjugates containing vancomycin molecules linked to each terminalthrough the vancomycin N-methyl amino group.

Further, in certain aspects of the invention, a low molecular weight(e.g. less than about 10,000) releasable polymer residue, or smallmolecular weight protecting group is used to temporarily protect thesugar amino group (V₃) in order to prepare the selectivepolymer-vancomycin N-methyl amino (X₁) derivatives. These protectinggroups can be removed once the X₁ amino group(s) has/have beenderivatized. The protecting groups can be hydrolyzed either in vitro ina PBS or similar buffer followed by purification or in vivo based uponenzymatic degradation.

In yet another aspect of the invention, polymer-vancomycin conjugateshaving a polymer residue attached on both the sugar amino and theN-methyl amino of said vancomycin compound are prepared by the reactionof the V₃-linked vancomycin compound with a second activated polymerlinker in the presence of at least about a 5 fold molar excess amount ofdimethylaminopyridine (DMAP) and a sufficient amount of a solvent whichcontains a mixture of dichloromethane (DCM) and dimethylformamide (DMF).In preferred aspects, the amount of base is from about 2 to about 20fold molar excess and in more preferred aspects, the amount of base isfrom about 5 to about 10 fold molar excess. The solvent mixture ispreferably about equal parts dichloromethane and dimethylformamidealthough the ratio of solvents can range from about 3:1 to about 1:3.

This alternative method provides the artisan with a more direct path toproviding vancomycin-compounds containing identical polymer residuesattached to both the V₃ and X₁ amino groups.

The activated polymers which can be employed in this process arepreferably selected from among the linear, terminally branched andmulti-arm polymer residues such as those described herein above.

Preferably the substituents are reacted in an inert solvent such asdimethylformamide (DMF), methylene chloride (DCM), tetrahydrofuran(THF), acetonitrile (CH₃CN), chloroform (CHCl₃), or mixtures thereof.The reaction is preferably conducted at a temperature from 0° C. up toabout 22° C. (room temperature).

Regardless of the route selected, some of the preferred compounds whichresult from the synthetic techniques described herein include:

wherein:

-   -   PEG is

-   -   (a) is an integer of from about 1 to about 5;    -   Z is O, NR₅, S, SO or SO₂; where R₈ is H, C₁₋₈ alkyl, C₁₋₈        branched alkyl, C₁₋₈ substituted alkyl, aryl or aralkyl;    -   (m) is 0 or 1;    -   (p) is a positive integer, preferably from about 1 to about 6;    -   x is 10 to 2,300;    -   Va is

and Vb is:

wherein, d, e, q, and s are each independently 0 or 1, and all othervariables are as described above.

E. Methods of Treatment

Another aspect of the present invention provides methods of treatmentfor various vancomycin-sensitive infections in mammals. The methodsinclude administering to the mammal in need of such treatment, aneffective amount of the prodrug, i.e. vancomycin, which has beenprepared as described herein.

The amount of the prodrug administered will depend upon the vancomycincompound selected. Generally, the amount of prodrug used in thetreatment methods is that amount which effectively achieves the desiredtherapeutic result in mammals. Naturally, the dosages of the variousprodrug compounds will vary somewhat depending upon the parent compound,rate of in vivo hydrolysis, molecular weight of the polymer, etc. Ingeneral, however, the vancomycin prodrugs are administered in amountsranging from about 0.5 to about 60 mg/kg twice a week. Preferably,vancomycin is administered in amounts ranging from about 0.5 to about 30mg/kg per day. The range set forth above is illustrative and thoseskilled in the art will determine the optimal dosing of the prodrugselected based on clinical experience and the treatment indication.Actual dosages will be apparent to the artisan without undueexperimentation.

The prodrugs of the present invention can be included in one or moresuitable pharmaceutical compositions for administration to mammals. Thepharmaceutical compositions may be in the form of a solution,suspension, tablet, capsule or the like, prepared according to methodswell known in the art. It is also contemplated that administration ofsuch compositions may be by the oral and/or parenteral routes dependingupon the needs of the artisan. A solution and/or suspension of thecomposition may be utilized, for example, as a carrier vehicle forinjection or infiltration of the composition by any art known methods,e.g., by intravenous, intramuscular, subdeimal injection and the like.

Such administration may also be by infusion into a body space or cavity,as well as by inhalation and/or intranasal routes. In preferred aspectsof the invention, however, the prodrugs are parenterally administered tomammals in need thereof.

Another aspect of the invention is a method of treating vancomycinsusceptible diseases in mammals using a combination of a vancomycin inunmodified or commonly available forms, e.g. vancomycin HCl, or otherpharmaceutically acceptable salt, solvate or hydrate thereof and apolymeric conjugate of the invention. The total amount of vancomycinadministered to the patient in need thereof is an effective amount asmentioned above, based on the amount of vancomycin. The combination ofprodrug and vancomycin derivative can be administered to a patient inneed of the drug or such treatment as part of a single pharmaceuticaldosage form (e.g. intravenous or parenteral injection/infusion or oraldosage form) or as part of a treatment regimen in which both of thevancomycin and vancomycin prodrug are administered as separate dosageforms to a patient in need thereof. Thus, the vancomycin and polymericconjugate of the invention are administered either substantiallyconcurrently in separate dosage forms or combined in a unit dosage form.

Since the present invention can relate to treatment with a combinationof vancomycin dosage forms which can be administered separately, theinvention also relates to combining separate pharmaceutical compositionsin kit form. That is, a kit is contemplated wherein two separate unitdosage forms are combined: for example a vancomycin pharmaceuticalcomposition and a separate pharmaceutical composition containing apolymer conjugate of the invention. The kit will preferably includedirections for the administration of the separate components. The kitform is particularly advantageous when the separate components must beadministered in different dosage forms and/or are administered atdifferent dosage intervals. Thus a kit may comprise, in separatecontainers in a single package, pharmaceutical compositions for use in atherapeutically effective amount of vancomycin or a pharmaceuticallyacceptable salt solvate or hydrate thereof in a pharmaceuticallyacceptable carrier and in a second container a therapeutically effectiveamount of a polymer conjugate as described herein in the form of apharmaceutically acceptable salt, solvate or hydrate thereof in apharmaceutically acceptable carrier.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention. The underlined and bold-faced numbers recited belowcorrespond to those shown in FIGS. 1-5.

General Procedures. All reactions were run under an atmosphere of drynitrogen or argon. Commercial reagents were used without furtherpurification. All PEG compounds were dried under vacuum or by azeotropicdistillation from toluene prior to use. ¹³C NMR spectra were obtained at75.46 MHz using a Varian Mercury 300 NMR spectrometer and deuteratedchloroform and pyridine as the solvents unless otherwise specified.Chemical shifts (δ) are reported in parts per million (ppm) downfieldfrom tetramethylsilane (TMS).

All vancomycin-polymer conjugation reactions were carried out in thepresence of 4 Angstrom molecular sieves.

HPLC method. The reaction mixtures and the purity of intermediates andfinal products were monitored by a Beckman Coulter System Gold® HPLCinstrument. It employs a ZOBAX® 300SB C8 reversed phase column (150×4.6mm) or a Phenomenex Jupiter® 300A C18 reversed phase column (150×4.6 mm)with a multiwavelength UV detector, using a gradient of 10-90% ofacetonitrile in 0.05% trifluoroacetic acid (TFA) at a flow rate of 1mL/min.

Example 1

Compound 4. A solution of 2 (mw 40 kDa, 23 g, 0.575 mmol) anddisuccinimidyl carbonate (DSC, 2.36 g, 9.2 mmol) in methylene chloride(DCM, 230 mL) and dimethylformamide (DMF, 23 mL) was cooled to 0° C.,followed by the addition of pyridine (0.75 mL, 9.2 mmol). This mixturewas allowed to warm to room temperature overnight, followed byfiltration through Celite® and partial removal of the solvent from thefiltrate by rotary evaporation under reduced pressure. The crude productwas precipitated with ether and collected by filtration, andcrystallized from 20% DMF/isopropanol (IPA) to yield 4 (20.1 g, 0.496mmol, 86%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 168.2, 151.1, 70.7-69.6 (PEG),68.0, 45.2, 25.2.

Example 2

Compound 5. To a solution of 1 (0.550 g, 0.37 mmol) and triethylamine(TEA, 2.06 mL, 14.8 mmol) in DMF (50 mL) was added 4 (3 g, 0.074 mmol)and 5.5 g molecular sieves (4 Å) and the mixture stirred at 30° C. for 5hrs. The reaction mixture was filtered through celite, the PEG conjugateprecipitated with ether, filtered, and crystallized from DMF/ethanol(50:50) three times to give 5 (2.0 g, 0.0436 mmol, 59%).

Example 3

Compound 6. A solution of 2 (10 g, 0.025 mmol) in toluene (150 mL) wasazeotroped for 2 hrs with the removal of 50 mL of distillate. Thereaction mixture was then cooled to 30° C., followed by the addition ofa 1.0 M solution potassium t-butoxide in t-butanol (1.7 ml, 1.7 mmol).The resulting mixture was stirred for 1 hr at 45° C., cooled to 30° C.,followed by the addition of t-butyl bromoacetate (0.4 g, 2.0 mmol). Theresulting mixture was refluxed for 18 hrs, followed by filtrationthrough Celite and partial removal of the solvent from the filtrate byrotary evaporation under reduced pressure. The crude product wasprecipitated with ether, collected by filtration, and crystallized fromIPA to yield 6 (8.1 g, 0.20 mmol, 80%). ¹³C NMR (67.8 MHz, C₅D₅N) δ169.1, 81.1, 72.2-69.6 (PEG), 68.7, 45.2, 27.9.

Example 4

Compound 7. A solution of 6 (10.6 g, 0.26 mmol) in DCM (100 mL) andtrifluoroacetic acid (TEA, 50 mL) was stirred for 7 hrs at roomtemperature, followed by partial removal of DCM by evaporation underreduced pressure. The product was precipitated with ethyl ether,filtered, and washed with ether to yield 7 (9.3 g, 0.0178 mmol, 89%).¹³C NMR (67.8 MHz, C₅D₅N) δ 170.9, 72.2-69.6 (PEG), 68.2, 45.2.

Example 5

Compound 8. A solution of 7 (11 g, 0.273 mmol), N-hydroxysuccinimide(NHS, 0.25 g, 2.19 mmol), and diisopropylethyl amine (DIEA, 1.1 mL, 6.56mmol) in DCM (88 mL) and DMF (22 mL) was cooled to 0° C., followed bythe addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC, 0.63 g, 3.28 mmol). This mixture was allowed to warmto room temperature overnight, followed by filtration through Celite®and partial removal of the solvent from the filtrate under reducedpressure. The crude product was precipitated with ether, collected byfiltration and crystallized from 20% DMF/IPA to yield 8 (10.4 g, 0.257mmol, 94%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 168.3, 165.5, 72.2-68.3 (PEG),66.2, 45.2, 25.3.

Example 6

Compound 9. To a solution of 1 (0.66 g, 0.44 mmol) and TEA (2.47 mL,17.77 mmol) in DMF (60 mL) was added 8 (3 g, 0.074 mmol) and 6.6 gmolecular sieves (4 Å), and the resulting mixture stirred at 30° C. for12 hrs. The reaction mixture was filtered through celite, the PEGconjugate precipitated with ether, and finally crystallized fromDMF/ethanol (50:50) three times to give 9 (2.4 g, 0.0522 mmol, 71%).

Example 7

Compound 10. A solution of 4 (6.86 g, 0.169 mmol) and glycinet-butylester (0.227 g, 1.36 mmol) in DCM (70 mL) wag cooled to 0° C.,followed by the addition of DMAP (0.165 g, 1.36 mmol). The mixture wasallowed to warm to room temperature overnight, followed by filtrationand partial removal of the solvent from the filtrate by rotaryevaporator under reduced pressure. The crude product was precipitatedwith ether, collected by filtration, and crystallized from IPA to yield10 (6.11 g, 0.150 mmol, 89%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 168.7, 156.0,81.3, 72.19-70.3 (PEG), 69.2, 63.9, 45.3, 43.1, 27.9.

Example 8

Compound 11. A solution of 10 (6 g, 0.147 mmol) in DCM (60 mL) and TFA(30 mL) was stirred for 4 hrs at room temperature, followed by partialremoval of the solvents under reduced pressure. The product wasprecipitated with ethyl ether, filtered, and washed with ether to yield11 (5 g, 0.122 mmol, 83%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 170.5, 156.0,72.21-69.9 (PEG), 69.2, 63.8, 45.3, 42.2.

Example 9

Compound 12. A solution of 11 (2.5 g, 0.0619 mmol) and NHS (0.0569 g,0.495 mmol) in DCM (20 mL) and DMF (5 mL) was cooled to 0° C., followedby the addition of EDC (0.143 g, 0.743 mmol) and DIEA (0.26 mL, 1.485mmol). This mixture was allowed to warm to room temperature overnight,followed by filtration and partial removal of the solvent from thefiltrate by evaporation in vacuo. The crude product was precipitatedwith ether, filtered, and crystallized from 20% DMF/IPA to give 12 (2.1g, 0.0520 mmol, 84%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 168.5, 165.9, 156.1,72.21-70.3 (PEG), 69.9, 64.2, 45.3, 40.3, 25.4.

Example 10

Compound 13. To a solution of 1 (0.466 g, 0.314 mmol) and TEA (1.75 mL,12.5 mmol) in DMF (50 mL) was added (12) 8 (2.54 g, 0.0627 mmol) and 5 gmolecular sieves (4 Å). The mixture was stirred at 25-30° C. overnight,followed by filtration and precipitation with ether. The crude productwas crystallized from DMT/ethanol (1:1) to yield 13 (1.4 g, 0.0303 mmol,48%).

Example 11

Compound 15. A solution of 14 (10 g, 0.248 mmol) and aminoethoxy ethanol(0.5 mL, 4.96 mmol) in DCM (200 mL) was cooled to 0° C., followed by theaddition of EDC (0.952 g, 4.96 mmol) and DMAP (0.605 g, 4.96 mmol). Thereaction mixture was allowed to warm to room temperature overnight,followed by filtration and partial removal of the solvent by evaporationin vacuo. The crude product was precipitated with ether, filtered, andcrystallized from IPA to yield 15 (8.92 g, 0.219 mmol, 88% yield). ¹³CNMR (67.8 MHz, C₅D₅N) 170.9, 170.6, 155.8, 70.3-68.8 (PEG), 64.1, 61.3,51.5, 39.3, 38.0.

Example 12

Compound 16. A solution of 15 (5 g, 0.121 mmol) and DSC (0.494 g, 1.93mmol) in DCM (50 mL) and DMF (5 mL) was cooled to 0° C., followed by theaddition of pyridine (0.16 mL, 1.928 mmol). The mixture was allowed towarm to room temperature overnight, followed by filtration and partialremoval of the solvent by evaporation under reduced pressure. The crudeproduct was precipitated with ether, filtered, and crystallized from IPAto yielded 16 (4.2 g, 0.097 mmol, 80%). ¹³C NMR (67.8 MHz, C₅D₅N) δ171.0, 170.6, 169.1, 156.0, 151.6, 71.6-70.0 (PEG), 68.3, 64.4, 51.9,39.5, 38.1, 25.7.

Example 13

Compound 17. To a solution of 1 (0.465 g, 0.313 mmol) and TEA (1.75 mL,12.51 mmol) in DMF (50 mL) was added 16 (2.58 g, 0.0625 mmol) and 7 gmolecular sieves (4 Å), and the resulting mixture stirred at 25-30° C.overnight, followed by filtration. The crude product was precipitatedwith ether, filtered, and crystallized from DMF/ethanol (1:1) to give 17(2.1 g, 0.045 mmol, 72.1%).

Example 14

Compound 18. A solution of 3 (mw 20 kDa, 25 g, 1.25 mmol) and DSC (10.2g, 40.0 mmol) in methylene chloride (DCM, 250 mL) and dimethylforamide(DMF 25 mL) was cooled to 0° C., followed by the addition of pyridine(3.20 mL, 40.0 mmol). This mixture was allowed to warm to roomtemperature overnight, followed by filtration through Celite and partialremoval of the solvent from the filtrate by rotary evaporation underreduced pressure. The crude product was precipitated out with ether andcollected by filtration, crystallized from 20% DMF/IPA to yield 18 (23.4g, 1.11 mmol, 89%). ¹³C NMR (67.8 MHz, C₅D₅N) 168.2, 151.2, 70.5-69.5(PEG), 68.0, 25.2.

Example 15

Compound 19. To a solution of 1 (2.53 g, 1.70 mmol) and TEA (14.2 mL,102 mmol) in DMF (200 mL) was added 18 (3 g, 0.142 mmol) and 5.5 gmolecular sieves (4 Å) and the mixture stirred at 30° C. for 12 hrs. Thereaction mixture was filtered through celite, the PEG conjugateprecipitated with ether and purified by column chromatograph using gelfiltration column (superdex75 or sephadex G75) to give 19 (1.4 g, 0.044mmol, 59%) after lipholization. (Note: the product can also be purifiedby dialysis using a membrane with a molecular weight cut-off of12000-14000.)

Example 16

Compound 20. A solution of 3 (30 g, 1.50 mmol) in toluene (350 mL) wasazeotroped for 2 hrs with the removal of 50 mL of distillate. Thereaction mixture was then cooled to 30° C., followed by the addition ofa 1.0 molar solution potassium t-butoxide in t-butanol (24 ml, 24 mmol).The resulting mixture was stirred for 1 hr at 45° C., cooled to 30° C.,followed by the addition of t-butyl bromoacetate (9.4 g, 48.2 mmol). Theresulting mixture was refluxed for 18 hrs, filtered through Celite andthe solvent was concentrated under reduced pressure. The crude productwas precipitated with ether and collected by filtration, thencrystallized from IPA to yield 20 (26.2 g, 1.25 mmol, 83%). ¹³C NMR(67.8 MHz, C₅D₅N) δ 169.1, 81.1, 70.6-69.5 (PEG), 68.7, 27.9.

Example 17

Compound 21. A solution of 20 (13.0 g, 0.62 mmol) in DCM (130 mL) andTFA (65 mL) was stirred for 7 hrs at room temperature, followed bypartial removal of DCM by evaporation under reduced pressure. Theproduct was precipitated with ethyl ether, filtered, and washed withether to yield 21 (12.4 g, 0.61 mmol, 98%). ¹³C NMR (67.8 MHz, C₅D₅N) δ171.1, 71.3-69.5 (PEG), 68.3.

Example 18

Compound 22. A solution of 21 (1.5 g, 0.073 mmol), NHS (0.14 g, 1.2mmol), and DIEA (0.41 mL, 2.34 mmol) in DCM (12 mL) and DMF (3 mL) wascooled to 0° C., followed by the addition of ED C (0.34 g, 1.76 mmol).This mixture was allowed to warm to room temperature overnight, followedby filtration through Celite and partial removal of the solvent from thefiltrate under reduced pressure. The product was precipitated withether, collected by filtration, and crystallized from 20% DMF/IPA toyield 22 (1.2 g, 0.055 mmol, 75%). ¹³C NMR (67.8 MHz, C₅D₅N) δ 168.4,165.6, 71.0-69.5 (PEG), 25.4.

Example 19

Compound 23. To a solution of 1 (0.825 g, 0.55 mmol) and TEA (3.09 mL,22.2 mmol) in DMF (60 mL) is added 22 (1.2 g, 0.055 mmol) and 6.6 gmolecular sieves (4 Å) and the mixture stirred at 30° C. for 12 hrs. Thereaction mixture is filtered through celite, the PEG conjugateprecipitated with ether, and purified by column chromatograph using agel filtration column (superdex75 or sephadex G75) to give 23 (1.05 g,0.033 mmol, 60%) after lipholization. (Note: this product can also bepurified by dialysis using a membrane with a molecular weight cut-off of12000-14000.)

Other embodiments of the invention will be apparent to one skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

1. A compound of the formula (I)

wherein: R₃-R₅ are each independently selected from among hydrogen, C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ alkenyls, C₃₋₁₂ branched alkenyls, C₁₋₆ alkynyls, C₃₋₁₂branched alkynyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ hetero-alkyls,C₁₋₆ alkoxyalkyl, phenoxyalkyl and C₁₋₆ heteroalkoxys; R₆ is OH,NH-aryl, NH-aralkyl, or NH—C₁₋₁₂ alkyl; w is 1 or 2; Q_(a) is H or

wherein: R₁ is a polyalkylene oxide; L₁ is a hydrolysis resistantbifunctional linker; Y₁ is O, S or NR₅; q is 0 or a positive integer;and d is 0 or
 1. Q_(b) is H or

wherein: R₂ is a polyalkylene oxide; L₂ is a hydrolysis resistantbifunctional linker; Y₂ is O, S or NR₅; s is 0 or a positive integer;and e is 0 or
 1. 2. The compound of claim 1, wherein R₁ furthercomprises a capping group J selected from the group consisting of OH,NH₂, SH, CO₂H and C₁₋₆ alkyl moieties.
 3. The compound of claim 1wherein R₂ further comprises a capping group J selected from the groupconsisting of OH, NH₂, SH, CO₂H and C₁₋₆ alkyl moieties.
 4. The compoundof claim 1, wherein: Q_(a) is

Q_(b) is


5. The compound of claim 1 wherein: Y₁ and Y₂ are independently O; R₃and R₄ are each independently hydrogen or CH₃; R₆ is OH or NH-aryl; qand s are independently 0-2; and w is
 1. 6. L₁₋₂ are independentlyselected from the group consisting of amino acids and—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)—,—[C(O)]_(v)(CR₂₆R₂₇)_(t)—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇O)_(t)—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇O)_(t)(CR₂₈R₂₉)_(y)O—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇O)_(t)(CR₂₈R₂₉)_(y)—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)O—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)(CR₂₈CR₂₉O)_(y)NR₃₀—,—[C(O)]_(v)O(CR₂₆R₂₇)_(t)NR₃₀—,—[C(O)]_(v)O(CR₂₆R₂₇)_(t)O—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)NR₃₀—,—[C(O)]_(v)NR₂₅(CR₂₆R₂₇)_(t)(CR₂₈CR₂₉O)_(y)—,—[C(O)]_(v)NR₂₅(CR₂₆CR₂₇O)_(t)(CR₂₈R₂₉)_(y)NR₃₀—,—[C(O)]_(v)O(CR₂₆CR₂₇O)_(t)NR₃₀—,

wherein: R₂₅-R₃₀ are independently selected from the group consisting ofhydrogen, C₁₋₆ alkyls, C₂₋₆ alkenyls, C₂₋₆ alkynyls, C₃₋₁₉ branchedalkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₂₋₆ substitutedalkenyls, C₂₋₆ substituted alkynyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆hetero-alkyls, C₁₋₆ alkoxyalkyl, phenoxyalkyl and C₁₋₆ heteroalkoxys;R₃₁ is selected from the group consisting of hydrogen, C₁₋₆ alkyls, C₂₋₆alkenyls, C₂₋₆ alkynyls, C₃₋₁₉ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₂₋₆ substituted alkenyls, C₂₋₆ substitutedalkynyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆alkoxyalkyl, phenoxyalkyl and C₁₋₆ heteroalkoxys, NO₂, haloalkyl andhalogen; t and y are individually selected positive integers; and v is 0or 1; provided that Q_(a) and Q_(b) are both not simultaneously H. 7.The compound of claim 6, wherein the amino acid is selected from thegroup consisting of alanine, valine, leucine, isoleucine, glycine,serine, threonine, methionine, cysteine, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, lysine, arginine, histidineand proline.
 8. The compound of claim 1, wherein said polyalkylene oxidecomprises polyethylene glycol.
 9. The compound of claim 8, wherein saidpolyalkylene oxide is selected from the group consisting of:A-O—(CH₂CH₂O)_(x)—A-O—(CH₂CH₂O)—CH₂C(O)—O—,A-O—(CH₂CH₂O)_(x)—CH₂CH₂NR₇—,A-O—(CH₂CH₂O)_(x)—CH₂CH₂SH, wherein: A is a capping group selected fromthe group consisting of OH, NH₂, SH, CO₂H and C₁₋₆ alkyl moieties; and xis an integer of from about 10 to about 2,300.
 10. The compound of claim1, wherein said polyalkylene oxide has a total number average molecularweight of from about 5,000 to about 100,000 daltons.
 11. The compound ofclaim 10, wherein said polyalkylene oxide has a total number averagemolecular weight of from about 10,000 to about 80,000 daltons.
 12. Thecompound of claim 10, wherein said polyalkylene oxide has a total numberaverage molecular weight of from about 20,000 to about 40,000 daltons.13. A compound of claim 1, wherein: Q_(a) is

wherein: R₁ is A-O—(CH₂CH₂O)_(x)—CH₂C(O)—O—, wherein A is a cappinggroup selected from the group consisting of OH, NH₂, SH, CO₂H and C₁₋₆alkyl moieties and x is an integer of from about 10 to about 2,300; Y₁is O q is 0-2; d is 0 or 1; Q_(b) is hydrogen; R₃ and R₄ are eachindependently hydrogen or CH₃, R₆ is OH or NH-aryl; and w is
 1. 14. Acompound of claim 1, wherein: Q_(b) is

wherein: R₂ is A-O—(CH₂CH₂O)_(x)—CH₂C(O)—O—, wherein A is a cappinggroup selected from the group consisting of OH, NH₂, SH, CO₂H and C₁₋₆alkyl moieties and x is an integer of from about 10 to about 2,300; Y₂is O; s is 0-2; e is 0 or 1; Q_(a) is hydrogen; R₃ and R₄ are eachindependently hydrogen or CH₃; R₆ is OH or NH-aryl- and w is
 1. 15. Aprocess for preparing a compound of claim 1 comprising, reacting avancomycin compound of the formula:

wherein R₃ and R₄ are independently selected from the group consistingof hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₃₋₈ substituted cycloalkyls, aryls, substitutedaryls, aralkyls, C₁₋₆ hetero-alkyls, substituted C₁₋₆ hetero-alkyls,C₁₋₆ alkoxyalkyl, phenoxyalkyl and C₁₋₆ heteroalkoxys; R₆ is OH,NH-aryl, NH-aralkyl, or NH—C₁₋₁₂ alkyl; and w is 1 or 2; with a polymerresidue containing at least one leaving group capable of reacting withthe sugar amino group of said vancomycin compound in the presence of atleast about a twenty-fold molar excess of triethylamine and a sufficientamount of dimethylformamide.
 16. The process of claim 15, furthercomprising reacting said sugar amino compound with a second activatedpolymer residue containing at least one leaving group capable ofreacting with the N-methyl-amino group of said conjugate in the presenceof at least about a 5 fold molar excess of dimethylaminopyridine and asufficient amount of a solvent mixture of dichloromethane anddimethylformamide.
 17. The process of claim 16, wherein said solventmixture comprises about equal parts dichloromethane anddimethylformamide.
 18. A method of treating a bacterial infection in amammal comprising administering an effective amount of a compound ofclaim 1, to a mammal in need of such treatment, whereby the compound ofclaim 1 undergoes degradation and releases vancomycin in vivo.
 19. Amethod of treating a bacterial infection in a mammal comprisingadministering an effective amount of a compound of claim 13, to a mammalin need of such treatment, whereby the compound of claim 13 undergoesdegradation and releases vancomycin in vivo.
 20. A method of treating abacterial infection in a mammal comprising administering an effectiveamount of a compound of claim 14, to a mammal in need of such treatment,whereby the compound of claim 14 undergoes degradation and releasesvancomycin in vivo.
 21. A method of treating a bacterial infection in amammal comprising administering to a mammal in need of such treatment,an effective amount of a combination of vancomycin or a pharmaceuticallyacceptable salt thereof and a compound of claim
 1. 22. A kit comprisingin separate containers in a single package, pharmaceutical compositionsfor use in combination to treat a bacterial infection which comprises inone container a therapeutically effective amount of vancomycin or apharmaceutically acceptable salt thereof in a pharmaceuticallyacceptable carrier and in a second container a therapeutically effectiveamount of a compound of claim 1 or a pharmaceutically acceptable saltthereof in a pharmaceutically acceptable carrier.